Glucose isomerase (E.C.5.3.1.5 or xylose isomerase) is a key enzyme in the pentose phosphate pathway. It is one of the most important industrial enzymes (Kaneko et al., Bioscience, Biotechnology, and Biochemistry 2000, 64:940-947). In bio-energy industry, people are trying to use the enzyme, together with other key enzymes in the degradation pathway of cellulose and hemicelluloses, to produce bioethanol commercially. For example, the pathway of the enzymatic degradation of xylan to xylulose-5-phosphate is as follows: xylan is converted into xylo-oligosaccharides in the presence of β-1,4-xylanase, xylo-oligosaccharides is further turned into xylose by β-xylosidase. Then, xylose is converted to xylulose by glucose isomerase (xylose isomerase) and xylulose is converted to xylulose-5-phosphate by β-xylulokinase.
Under the pressure of environmental pollution and the shortage of oil, many countries in the world, such as Brazil, the United States and Canada have been using sugar cane or corn as starting materials to produce fuel ethanol. China has also been using corn or other crops to produce bioethanol. As biomass is very rich in the world, scientists in different countries are exploring ways to use cellulose- and hemicellulose-rich agricultural residues to produce bioethanol industrially. Glucose isomerase is a key enzyme in the biodegradation of hemicellulose. With high activity, or with both of high activity and thermostability glucose isomerase can efficiently reduce the cost of bioethanol production to facilitate industrial production of bioethanol.
Scientists around the world have been working on the identification of thermostable and highly active glucose isomerases from thermophilic bacteria, and production of the same via protein engineering. J. G. Zeikus and his collaborators isolated and studied thermostable glucose isomerases from thermophilic bacteria, such as Thermoanaerobacterium saccharolyticum and Thermotoga neapolitana (Lee et al., Journal of General Microbiology, 139:1227-1234, 1993; Vieille et al., Methods in Enzymology, 330:215-24, 2001; Lee et al., Journal of General Microbiology, 139:1241-1243, 1993; Scriprapundh et al., Protein Engineering, 13:259-265, 2000; Scriprapundh et al., Protein Engineering, 16:683-690, 2003; Zeikus et al., U.S. Pat. No. 5,656,497). Nevertheless, the thermostabilities of the thermostable glucose isomerases from these and other bio-resources are still much to be desired as the activities thereof are low, and thus are not applicable to industrial applications. Therefore, glucose isomerase with high activity, or high activity and thermostability remains desirable.